When animals are bred in an industrial environment, it is desirable that the animals have predetermined or previously estimated qualities. As a consequence, there is a continuous need to improve the efficiency of breeding methods for the animals in qualitative and quantitative aspects. For instance, in cows where breeding is limited at the level of the individual animals and in pigs where breeding is limited at the level of selection lines, the improved breeding methods allows for increased efficiency of the predetermined or previously estimated qualities.
One breeding method known in the art is artificial insemination (“AI”). With artificial insemination, an ejaculate is obtained from a male animal. Portions of the ejaculate can be inseminated as obtained, can be treated to improve the quality of the ejaculate, or more doses may be obtained from the male animal for insemination. When the sperm within the ejaculate is treated, the seed is analyzed, diluted in a suitable medium such as a BTS-medium, and further treated using methods known in the art to provide a suitable sample that can be used as an inseminate.
By pre-treating and/or diluting the ejaculate, it is possible to obtain a large number of doses from the ejaculate wherein each dose can be used for insemination. For instance, 20-40 doses suitable for artificial insemination can be obtained from one pig ejaculate, wherein each a dose generally contains about 2.5 billion sperm cells. The current methods used for the artificial insemination of pigs results in fertility results of about 90% and about 11 piglets per litter.
Although these results are acceptable, it is desirable to obtain a smaller insemination dose that does not have a negative effect on the fertility result and/or the litter size. This would lead to more homogeneous animal populations and more uniform products. A more efficient insemination dose would also lead to a more efficient use of the sperm of animals and result in substantial economic advantages. Other benefits of a more efficient insemination dose include increased genetic gains in nucleus breeding such as a larger number of progeny, an increased rate of dissemination of superior traits into a population, an increased efficiency of reproduction, i.e., larger litter sizes or higher farrowing rates, and less sperm needed per insemination. The use of less sperm can be cheaper to produce since diluted semen is less expensive than more concentrated semen. The latter advantage could stimulate the use of frozen semen for routine AI, which has important veterinary health advantages (i.e., prevention of diseases).
However, one of the disadvantages of insemination, whether through natural or artificial routes, is that within hours after the insemination, the number of sperm cells in the female genital tract is dramatically reduced. The reduction in the number of sperm cells is ascribed to phagocytosis of the sperm cells by uterine leukocytes, such as Polymorphonuclear Neutrophils (PMN). Vast numbers of these PMNs are recruited to the lumen of the uterus shortly after insemination. The result is that a large amount of the inseminated sperm is not used. For instance, it has been found that after 8 hours, only about 1% of the amount of the originally artificially inseminated sperm remains and after 24 hours, only about 0.1% of the amount of inseminated sperm remains.
The phagocytosis of the sperm cells by the PMNs is why many sperm cells are needed per insemination. Moreover, the rapidly declining sperm numbers also leads to a lower fertility rate, and hence is a disadvantage of the known insemination methods. This is even more disadvantageous since the exact time of ovulation is difficult to estimate, especially with pigs. The negative effect of inseminating too early with respect to ovulation cannot be “repaired” by increasing the sperm dosage, even to as much as 6 billion sperm per dose (Steverink et al., J. Reprod. Fert. 1997, 111, 165-171) since more sperm may simply elicit more PMN recruitment and phagocytosis activity.
Thus, the combination of increased phagocytosis and the uncertainty of the exact time of ovulation leads to an even lower fertility rate. Even if the exact time of ovulation and consequently the time of insemination could be determined adequately, insemination at the right time is still of critical importance for a good fertility rate. Thus, the relatively fast phagocytosis of the sperm is still a disadvantage of the presently known insemination techniques.
There also exists a need for the improvement of fertilization techniques with respect to human in vitro fertilization. With human in vitro fertilization, it is known that improved fertilization results can be obtained by capacitation of the sperm. Capacitation is connected with a large amount of specific cellular changes in the sperm related to the ability of the sperm to the fertilization the oocyte.
Caffeine may be used to accelerate certain phases of capacitation. Caffeine stimulates the motility of sperm and aids in inducing the state of the so called “hyperactivated motility,” which is seen as a specific stage of capacitation. Due to the increased motility of the sperm, the in vitro fertility rate is increased even though the lifespan of the sperm is reduced.
A major disadvantage of the capacitation of sperm is that after capacitation, the sperm has a shorter lifespan which reduces the period of time available for fertilization, or the period of time from the moment of insemination until the moment at which the sperm is no longer fertile. Thus, the use of capacitation techniques on sperm for artificial insemination with animals, such as mammals, is desirable. However, capacitation is not suitable with pigs and birds because of the shortened life expectancy of the sperm in the animals. The use of capacitation in pigs is further disadvantageous since determining the time of ovulation in pigs is difficult.